Methotrexate (MTX) inhibition of cytokine production: relationship with clinical outcome and genetic polymorphisms

Introduction

Downregulation of TNF-α levels in synovial fluid of rheumatoid arthritis (RA) has been described following treatment with methotrexate (MTX). Although the mechanism of action of MTX is unknown, inhibition of methylenetetrahydrofolate reductase (MTHFR) could be implicated. Polymorphisms in MHTFR have been described (A1298T and C677T) that may condition clinical response.

Aim

To determine whether MHTFR genotype is related to the 'in vitro' cytokine inhibition and 'in vivo' clinical response to MTX.

Materials and methods

Twenty-three patients with early RA were studies along with 23 healthy donors. Blood (1/10 diluted in LPS-free Iscove’s) was cultured in the presence of 1 μg/ml anti-CD3 and 1 μg/ml anti-CD28, with or without MTX (0.152 to 625 ng/ml; L. Aarden, personal communication). The effect of MTX was reverted with folinic acid (3 μg/ml). TNF-α, IFN-γ and IL-6 concentrations weremeasured by ELISA in sera and culture supernatants after 72 hours. MTHFR polymorphisms were determined by PCR-RFLP. DAS was used to evaluate the clinical effect of MTX at 6 months.

Results

1) Basal and stimulated cytokine production was similar in controls and patients. 2) 40 ng/ml of MTX significantly inhibited IFN-γ, TNF-α and IL-6 production of T cells stimulated by anti-CD3+anti-CD28 (P < 0.001). 3) A statistically significant correlation (P < 0.05) was found between ID-50 for TNF-α and clinical improvement as assessed by DAS (% decrease of DAS score). ID-50 for TNF-α was not related to MHTFR polymorphisms (P = 0.076). Median ID-50 was 27.13 for TNF-α (25th percentile = 18.5; 75th percentile = 34.16) and 19.74 for IFN-γ (25th percentile = 13.08; 75th percentile = 24.00). 4) ID-50 for IFN-γ was lower in homozygous individuals A1298A (P < 05), but was not associated with clinical outcome. No statistical differences were associated to the C677T mutation.

Conclusions

MTX inhibits stimulated T cell cytokine production. Individual susceptibility for MTX inhibition of cytokine production could help predict clinical response to the drug. Mutations in the MHTFR gene were associated with a lower response to MTX 'in vitro'.